Coagulation factor VIIa is the key initiator of haemostasis. It is a 50-kDa plasma protein with a functional circulatory half life around 1-3 hours. The mature protein contains a total of 24 cysteine residues paired in 12 disulfide bondings. Of these, the disulfide bond bridging Cys340 and Cys368 in the protease domain is highly labile and readily reduced by even low concentrations of commonly used reducing agents such as β-mercaptoethanol, see Higashi (1997). Cys340-Cys368 (or Cys191-Cys220 according to chymotrypsin numbering) is highly conserved in the trypsin family of serine proteases including coagulation factors II, IX, X, XI and protein C, see Wang (1997). It forms part of the wall of the S1 binding pocket and in FVIIa reduction of the disulfide bond results in a loss of amidolytic activity, see Higashi (1997).
Covalent modification, e.g. by PEGylation or lipid attachment, has been successfully applied on several protein-based pharmaceutics to improve their pharmacokinetic and pharmacodynamic profiles. Conjugation via native or engineered cysteines provides an attractive means of site-specific modification due to the rarity of this amino acid on the surface of proteins, particularly those secreted by the cell, as well as the high selectivity of the thiol-coupling chemistry.
Native Factor VIIa contains 24 cysteine residues and disulfide bridges are established between the following cysteine residues: C17 and C22, C50 and C61, C55 and C70, C72 and C81, C91 and C102, C98 and C112, C114 and C127, C135 and C262, C159 and C164, C178 and C194, C310 and C329, and between C340 and C368.
The lack of free thiols in native Factor VIIa has led to the proposal that prolongation of the circulatory half life might be achieved by modification, e.g. PEGylation, of engineered solvent-exposed cysteines, see, e.g. WO 02/077218 A1 and WO 01/58935 A2.
Hence, WO 01/58935 A2 discloses Factor VII polypeptide conjugates with non-polypeptide moieties and their preparation. It is, i.a., suggested that the non-polypeptide moiety is conjugated to the Factor VII polypeptide via a cysteine. Similarly, WO 02/077218 A1 discloses Factor VII polypeptide conjugates with chemical groups and their preparation. It is, i.a., suggested that the chemical group is conjugated to the Factor VII polypeptide via a cysteine.
In practice, however, this approach is somewhat complicated by the fact that introduced cysteines in predominant portions of the Factor VII polypeptide are found as mixed disulfides with low-molecular weight thiol compounds such as glutathione (γ-glutamyl-cysteinylglycine), cysteine, and homocysteine (see FIG. 1) when the polypeptide is prepared by recombinant techniques, thereby preventing subsequent chemical conjugation via the thiol groups of the cysteines. Thus, there is a need for methods in which mixed disulfides of such cysteines and low-molecular weight thiols can be chemically reduced with preservation of the native disulfide bonds.
SE 9501285A discloses a process for the in vitro production of appropriately folded, biologically active disulfide-crosslinked proteins using a mixture of a protein disulfide oxidoreductase (e.g. protein disulfide isomerase (PDI)), a glutaredoxin and a redox buffer. The reference is focused on cysteines involved in intramolecular disulfide bonds.